This invention relates to novel protease inhibitors, particularly inhibitors of cysteine and serine proteases, more particularly compounds which inhibit cysteine proteases. The compounds of this invention even more particularly relate to those compounds which inhibit cysteine proteases of the papain superfamily, and particularly cysteine proteases of the cathepsin family. In the most preferred embodiment, this invention relates to compounds which inhibit cathepsin K. Such compounds are particularly useful for treating diseases in which cysteine proteases are implicated, especially diseases of excessive bone or cartilage loss, e.g., osteoporosis, periodontitis, and arthritis.
Cathepsin K is a member of the family of enzymes which are part of the papain superfamily of cysteine proteases. Cathepsins B, H, L, N and S have been described in the literature. Recently, cathepsin K polypeptide and the cDNA encoding such polypeptide were disclosed in U.S. Pat. No. 5,501,969 (called cathepsin O therein). Cathepsin K has been recently expressed, purified, and characterized. Bossard, M. J., et al., (1996) J. Biol. Chem. 271, 12517-12524; Drake, F. H., et al., (1996) J. Biol. Chem. 271, 12511-12516; Bromme, D., et al., (1996) J. Biol. Chem. 271, 2126-2132.
Cathepsin K has been variously denoted as cathepsin O, cathepsin X or cathepsin O2 in the literature. The designation cathepsin K is considered to be the more appropriate one (name assigned by Nomenclature Committee of the International Union of Biochemistry and Molecular Biology).
Cathepsins of the papain superfamily of cysteine proteases function in the normal physiological process of protein degradation in animals, including humans, e.g., in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cathepsins have been implicated in various disease states, including but not limited to, infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei brucei, and Crithidia fusiculata; as well as in schistosomiasis malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like. See International Publication Number WO 94/04172, published on Mar. 3, 1994, and references cited therein. See also European Patent Application EP 0 603 873 A1, and references cited therein. Two bacterial cysteine proteases from P. gingivallis, called gingipains, have been implicated in the pathogenesis of gingivitis. Potempa, J., et al. (1994) Perspectives in Drug Discovery and Design, 2, 445-458.
Cathepsin K is believed to play a causative role in diseases of excessive bone or cartilage loss. Bone is composed of a protein matrix in which spindle- or plate-shaped crystals of hydroxyapatite are incorporated. Type I Collagen represents the major structural protein of bone comprising approximately 90% of the structural protein. The remaining 10% of matrix is composed of a number of non-collagenous proteins, including osteocalcin, proteoglycans, osteopontin, osteonectin, thrombospondin, fibronectin, and bone sialoprotein. Skeletal bone undergoes remodeling at discrete foci throughout life. These foci, or remodeling units, undergo a cycle consisting of a bone resorption phase followed by a phase of bone replacement.
Bone resorption is carried out by osteoclasts, which are multinuclear cells of hematopoietic lineage. The osteoclasts adhere to the bone surface and form a tight sealing zone, followed by extensive membrane ruffling on their apical (i.e., resorbing) surface. This creates an enclosed extracellular compartment on the bone surface that is acidified by proton pumps in the ruffled membrane, and into which the osteoclast secretes proteolytic enzymes. The low pH of the compartment dissolves hydroxyapatite crystals at the bone surface, while the proteolytic enzymes digest the protein matrix. In this way, a resorption lacuna, or pit, is formed. At the end of this phase of the cycle, osteoblasts lay down a new protein matrix that is subsequently mineralized. In several disease states, such as osteoporosis and Paget""s disease, the normal balance between bone resorption and formation is disrupted, and there is a net loss of bone at each cycle. Ultimately, this leads to weakening of the bone and may result in increased fracture risk with minimal trauma.
The abundant selective expression of cathepsin K in osteoclasts strongly suggests that this enzyme is essential for bone resorption. Thus, selective inhibition of cathepsin K may provide an effective treatment for diseases of excessive bone loss, including, but not limited to, osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease. Cathepsin K levels have also been demonstrated to be elevated in chondroclasts of osteoarthritic synovium. Thus, selective inhibition of cathepsin K may also be useful for treating diseases of excessive cartilage or matrix degradation, including, but not limited to, osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix. Thus, selective inhibition of cathepsin K may also be useful for treating certain neoplastic diseases.
It now has been discovered that a novel class of compounds are protease inhibitors, most particularly inhibitors of cathepsin K, and these compounds are useful for treating diseases in which inhibition of bone resorption is indicated, such as osteoporosis and periodontal disease.
An object of the present invention is to provide protease inhibitors, such as inhibitors of cysteine and serine proteases. In particular, the present invention relates to compounds which inhibit cysteine proteases, and particularly cysteine proteases of the papain superfamily. Preferably, this invention relates to compounds which inhibit cysteine proteases of the cathepsin family and particularly, compounds which inhibit cathepsin K. The compounds of the present invention are useful for treating diseases, which may be therapeutically modified by altering the activity of such proteases.
Accordingly, in the first aspect, this invention provides a compound according to formula (I): 
In another aspect, this invention provides a pharmaceutical composition comprising a compound according to formula (I) and a pharmaceutically acceptable carrier.
In yet another aspect, this invention provides a method of treating diseases in which the disease pathology may be therapeutically modified by inhibiting proteases, such as cysteine and serine proteases. In particular, the method includes treating diseases by inhibiting cysteine proteases, and particularly cysteine proteases of the papain superfamily. More particularly, the inhibition of cysteine proteases of the cathepsin family, such as cathepsin K is described.
In another aspect, the compounds of this invention are especially useful for treating diseases characterized by bone loss, such as osteoporosis and gingival diseases, such as gingivitis and periodontitis, or by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
The present invention provides compounds of formula (I): 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3OC(O)NRxe2x80x2CH(R6)C(O);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each R5 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R6 is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
Rxe2x80x2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x3 is C1-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C2-6alkenyl or Het-C2-6alkenyl;
X is O or S; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof.
The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds of this invention. Prodrugs are any covalently bonded compounds which release the active parent drug according to formula (I) in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Inventive compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. According to the instant invention, the S-form at the furan ring junction of formula (I) compounds is preferred.
In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
The meaning of any substituent at any one occurrence in formula (I) or any subformula thereof is independent of its meaning, or any other substituent""s meaning, at any other occurrence, unless specified otherwise.
With respect to formula (I):
Suitably, R2 and R4 are H and R3 is C1-6alkyl or C2-6alkenyl. Preferably, R3 is i-butyl.
Suitably, each R5 is H.
Suitably, R1 is Rxe2x80x3OC(O), Rxe2x80x3SO2 or Rxe2x80x3C(O), in which Rxe2x80x3 is Ar-C0-6alkyl or Het-C0-6alkyl, and, most preferably, Rxe2x80x3 is 
in which B2 is OH, CN, OCF3, OC1-6alkyl, OAr, SO2C1-6alkyl, C1-6alkyl or halo.
Suitably, n is 1 or 2. Preferably, n is 1.
Suitably, X is O.
In one particular embodiment, this invention is a compound of formula (II): 
Preferably, the formula (I) compound of this invention is a compound of formula (IIa): 
Alternately, the formula (I) compound of this invention is a compound of formula (IIb): 
In another embodiment, this invention is a compound of formula (IIc): 
Specific representative compounds of this invention are:
4-(R,S)-Amino-N-[(3,4-methylenedioxybenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(3,4-dichlorobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(2-quinolinecarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(8-quinolinecarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-2,2-dibenzyl-tetrahydrofuran-3-one;
4-(R,S)-Amino-N[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(3,4-dimethoxybenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(indole-6-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(benzofuran-2-ylcarbonyl)-S-leucine)-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(5-aminobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(5-chlorobenzofuran-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(5-methoxybenzofuran-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(3-bromobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-bromobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(5-chlorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-fluorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-phenoxybenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4phenylbenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(6-trifluoromethylbenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-ethyllbenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-(tert-butyl)benzoyl)-S-leucine]-tetrahydrofuran-3-one ;
4-(R,S)-Amino-N-[(5-methoxybenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(4-nitrobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(6-bromobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(5-bromobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(6-methoxybenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(benzo(b)thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-R-Amino-N-[(benzo(b)thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(2-napthoyl)-S-leucine]-tetrahydrofuran-3-one;
4-R-Amino-N-[(2-napthoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(quinoline-2-carbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-R-Amino-N-[(quinoline-2-carbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(5-methoxybenzofuran-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[((4-pyrid-3-yl)benzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[((4-pyrid-2-yl)benzoyl)-S-leucine]-tetrahydrofuran-3-one,
4-S-Amino-N-[(benzyloxycarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(3,4-dimethoxybenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(benzofuran-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(4-[6-methylpyrid-3-yl]benzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(5-chlorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[((4-pyrid4-yl)benzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(2-chlorobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(4-bromobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(4-chlorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(4-benzylpiperidin-1-ylcarbonyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(3,4-dichlorobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-S-Amino-N-[(3-chlorobenzoyl)-S-leucine]-tetrahydrofuran-3-one;
4-(R,S)-Amino-N-[(3,4-dimethoxybenzoyl)-S-leucine]-tetrahydropyran-3-one;
4-(R,S)-Amino-N-[(4-phenoxybenzoyl)-S-leucine]-tetrahydropyran-3-one;
4-(R,S)-Amino-N-[(quinolin-2-ylcarbonyl)-S-leucine]-tetrahydropyran-3-one;
4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-tetrahydropyran-3-one:
4-(R,S)-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydropyran-3-one; and
4-(R,S)-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-tetrahydrothiophen-3-one;
or a pharmaceutically acceptable salt thereof.
In yet another aspect, this invention provides novel intermediates useful in the preparation of formula (I) compounds represented by formulae (III), (IV) and (V): 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3OC(O)NRxe2x80x2CH(R6)C(O);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each R5 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R6 is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
Rxe2x80x2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x3 is C1-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C2-6alkenyl or Het-C2-6alkenyl; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof, or 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3OC(O)NRxe2x80x2CH(R6)C(O);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each R5 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R6 is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
Rxe2x80x2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x3 is C1-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C2-6alkenyl or Het-C2-6alkenyl; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof, or 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3OC(O)NRxe2x80x2CH(R6)C(O);
R3 is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each R5 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R6 is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
Rxe2x80x2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x3 is C1-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C2-6alkenyl or Het-C2-6alkenyl;
X is O or S; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof.
Representative intermediates of this invention are:
trans-4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(tert-butoxycarbonyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-(S-leucine)-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(3,4-methylenedioxybenzoyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(3,4-dichlorobenzoyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(2-quinolinecarbonyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-3-hydroxytetrahydropyran;
trans-4-(R,S)-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-hydroxytetrahydropyran;
trans-4-(R,S)-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(indole-6-ylcarbonyl)-S-leucine]-3-hydroxytetrahydrofuran;
trans-4-(R,S)-Amino-N-[(5-aminobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-hydroxytetrahydrofuran
trans-4-Amino-3-hydroxytetrahydrofuran;
trans-3-Hydroxy-4-benzyloxycarbonylamino-tetrahydrofuran;
4-Benzyloxycarbonylamino-tetrahydrofuran-3-one;
3,3-Dimethoxy-4-benzyloxycarbonylamino-tetrahydrofuran;
3,3-Dimethoxy-4-amino-tetrahydrofuran
trans-4-S-Amino-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(benzyloxycarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-(S-leucine)-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(3,4-dichlorobenzoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(2-quinolinecarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran,
trans-4-S-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(benzofuran-2-ylcarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(2-naphthoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(5-methoxybenzofuran-2-ylcarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(5-chlorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(4-chlorobenzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(4-bromobenzoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(4-(pyrid-2-yl)benzoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S Amino-N-[(4-(pyrid-3-yl)benzoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-S-Amino-N-[(3,4-dimethoxybenzoyl)-S-leucine]-3-R-hydroxytetrahydrofuran;
trans-4-Amino-3-hydroxytetrahydropyran;
trans-4-(R,S)-Amino-N-[(benzyloxycarbonyl)-S-leucine]-3-hydroxytetrahydropyran;
trans-4-(R,S)-Amino-N-(S-leucine)-3-hydroxytetrahydropyran;
trans-4-(R,S)-Amino-N-[(benzo[b]thiophen-2-ylcarbonyl)-S-leucine]-3-hydroxytetrahydropyran;
N-benzo[b]thiophene-2-ylcarbonyl-L-leucine methyl ester;
N-benzo[b]thiophene-2-ylcarbonyl-L-leucine;
N-benzo[b]thiophene-2-ylcarbonyl-L-leucine-S-(methoxycarbonylmethyl)-L,D-cysteine ethyl ester; and
2-Methoxycarbonyl-4-(R,S)-Amino-N-[(benzo[b]thiophene-2-ylcarbonyl)-S-leucine]-tetrahydrothiophene-3-one;
or salts thereof.
These intermediates are prepared using methods analogous to that described in Schemes 1-4 and the Examples described hereinafter.
Prodrugs of compounds of the present invention may be a prodrug of the ketone functionality of formula (I) compounds, specifically ketals or hemiketals, of the formula (VI): 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3OC(O)NRxe2x80x2CH(R6)C(O);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each R5 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R6 is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
Rxe2x80x2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x3 is C1-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C2-6alkenyl or Het-C2-6alkenyl;
X is O or S;
n is 1, 2 or 3; and
Ra and Raxe2x80x2 independently are H or C1-2alkyl, with the proviso that when one of Ra and Raxe2x80x2 is H, the other is C1-2alkyl; or together are (CH2)2-3 forming a 5- or 6-membered ring;
or a pharmaceutically acceptable salt thereof.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984). The term xe2x80x9camino acidxe2x80x9d as used herein refers to the D- or L-isomers of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
xe2x80x9cC1-6alkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Any C1-6alkyl group may be optionally substituted independently by one or two halogens, SRxe2x80x2, ORxe2x80x2, N(Rxe2x80x2)2, C(O)N(Rxe2x80x2)2, carbamyl or C1-4alkyl, where Rxe2x80x2 is H or C1-6alkyl. C0alkyl means that no alkyl group is present in the moiety. Thus, Ar-C0alkyl is equivalent to Ar.
xe2x80x9cC3-6cycloalkylxe2x80x9d as applied herein is meant to include substituted (i.e., alkyl, OR, SR or halogen) and unsubstituted cyclopropane, cyclobutane, cyclopentane, and cyclohexane.
xe2x80x9cC2-6alkenylxe2x80x9d as applied herein means an alkyl group of 2 to 6 carbons,
wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond. C2-6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
xe2x80x9cC2-6alkynylxe2x80x9d means an alkyl group of 2 to 6 carbons, wherein one carbon-carbon single bond is replaced by a carbon-carbon triple bond. C2-6alkynyl includes acetylene, 1-propync, 2-propyne, 1-butyne, 2-butyne, 3-butyne, and the simple isomers of pentyne and hexyne.
xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d means F, Cl, Br, and I.
xe2x80x9cArxe2x80x9d or xe2x80x9carylxe2x80x9d means unsubstituted phenyl or naphthyl; or phenyl or naphthyl substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NRxe2x80x2Rxe2x80x2, O(CH2)1-6NRxe2x80x2Rxe2x80x2; wherein each Rxe2x80x2 independently is H, C1-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl; or phenyl or naphthyl substituted by one to three moieties selected from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2)2, F, Cl, Br and I, or substituted by a methylenedioxy group.
As used herein xe2x80x9cHetxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d represents a stable 5- to 7-membered monocyclic or a stable 7- to 10-membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or two moieties selected from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2)2, F, Cl, Br and I, where Rxe2x80x2 is as defined herein before. Examples of such heterocycles include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, thienyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, isothiazolyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothienyl, benzopyranyl, benzoxazolyl, benzofuranyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzoxazolyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, oxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzisoxazolyl, pyrimidinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,5-napthyridinyl, 1,6napthyridinyl, 1,7-napthyridinyl, 1,8-napthyridinyl, tetrazolyl, 1,2,3-triazolyl, and 1,2,4-triazolyl.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary butyl radical; Boc or BOC refers to the tbutyloxycarbonyl radical; Fmoc refers to the fluorenylmethoxycarbonyl radical; Ph refers to the phenyl radical; and Cbz or CBZ refers to the benzyloxycarbonyl radical.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide; EDC or EDCI refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide. HOBT or HOBt refers to 1-hydroxybenzotriazole; DMF refers to dimethyl formamide; DIEA refers to di-isopropylethylamine; Lawesson""s reagent is 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide; TFA refers to trifluoroacetic acid; and THF refers to tetrahydrofuran.
Compounds of the formula (I) are generally prepared by:
(i) reacting a compound of the formula (III): 
wherein R1, R2, R3, R4, R5 and n are as defined in formula (I), with any reactive functional groups protected, with an oxidizing agent; or
(ii) decarboxylating a compound of the formula (IV): 
wherein R1, R2, R3, R4, R5 and n are as defined in formula (I), with any reactive functional groups protected; or
(iii) reacting a compound of the formula (V): 
wherein R1, R3, R4, R5 and n are as defined in formula (I), with any reactive functional groups protected, with an acid;
and thereafter removing any protecting groups and optionally forming a pharmaceutically acceptable salt.
Compounds of the formula (I) are prepared by methods analogous to those described in the solution synthesis method of Scheme 1, or the solid support method of Scheme 2, or the solution synthesis method of Scheme 3. 
a) NaN3, ammonium chloride, methanol:water; b) 10% Pd/C, EtOH, H2; ethanolic HCl; c) trimethylacetyl chloride, N-BOC-leucine, DIEA, CH2Cl2; d) TFA, CH2Cl2; e) RCOCl, sodium hydrogen carbonate, 1,4-dioxane; f) Dess-Martin periodinane, CH2Cl2 
Compounds of the general formula (I), wherein n is 1 and R1 is Rxe2x80x3C(O) are prepared by methods shown in Scheme 1. Treatment of the known epoxide 1-Scheme-1 with sodium azide and ammonium chloride in aqueous methanol at elevated temperatures provides the azide 2-Scheme-1. Reduction of the azide 2-Scheme-1 utilizing methods that are known in the art, such as reduction with palladium on carbon in ethanol under an atmosphere of hydrogen, provides the amine salt 3-Scheme-1 after treatment with ethanolic hydrogen chloride. The amine salt 3-Scheme-1 may be coupled with a carboxylic acid by methods that are known in the art, such as acylation with an acid chloride or coupling with an acid in the presence of EDC and HOBT, to provide the amide 4-Scheme-1. The tert-butoxycarbonyl group may be removed by treatment with a strong acid, such as TFA, in an aprotic solvent, such as dichloromethane, to provide 5-Scheme-1. The salt 5-Scheme-1 may be acylated with an acid chloride in 1,4-dioxane in the presence of an aqueous base, such as saturated sodium hydrogen carbonate, to yield 6-Scheme-1. The alcohol 6-Scheme-1 may be oxidized by methods known in the art, such as by treatment with Dess-Martin periodinane, in an aprotic solvent, such as dichloromethane. 
Compounds of the general formula (I), wherein R1 is C(O)Rxe2x80x2, R2 is H, R4 is H, R5 is H, X is O, and n is 1, are prepared by the solid support synthesis (SPS) method shown in Scheme 2. In particular, in the first step, sodium triacetoxyborohydride is added to a stirred solution of Ellmans resin in DMF containing 1% HOAc, and then the xcex1-amino acid, methyl ester is added to give rise to 2-Scheme-2. The amine group is coupled with a carboxylic acid by using known methods, such as by the addition of the carboxylic acid with EDC, to give rise to the amide, 3-Scheme-2. Thereafter, the ester group is hydrolysed, for example, with potassium trimethylsilanoate in THF, and the liberated acid group is coupled with 3,3-dimethoxy-4-amino-tetrahydofuran using, for example, EDC in NMP, to give rise to 4-Scheme-2. The blocking group is then removed by known cleaving methods, such as by the addition of 7:2:1 TFA/CH2Cl2/H2O, to give rise to the desired compound, 5-Scheme-2.
Compounds of the general formula (I), wherein R1 is C(O)Rxe2x80x2, R2 is H, R4 is H, R5 is H, X is O, and n is 2, are prepared by the solid support synthesis (SPS) method shown in Scheme 2, except 3,3-dimethoxy-4-aminotetrahydropyran is substituted for 3,3-dimethoxy-4-amino-tetrahydofuran. 
(a) L-Leu methyl ester, iPr2NEt, CH2Cl2; (b) LiOH, THF/H2O; (c) (i)ClCO21Pr, Et3N, CH2Cl2 (ii) L-Cys ethyl ester (iii) BrCH2CO2Me; (d) (i)NaOMe, MeOH, (ii) AcOH/HCl, H2O
Scheme 4, hereinafter, shows the preparation of certain formula (I) compounds which are S-diastereomers at the furan ring junction. 
The R-diastereomer is prepared in an identical way from the opposite 3-azido4-hydroxytetrahydrofuran enantiomer: 
The intermediates of the present invention, such as 3,3-dimethoxy4-amninotetrahydofuran, can be prepared according to the method of Scheme 5. 
The steps in Scheme 5 are presented as follows. First, a nitrogen-protecting group is added by reacting 3-hydroxy4-aminotetrahydofuran with benzychloroformate in dioxane containing aqueous sodium carbonate. Thereafter, the alcohol group is oxidized by known methods, such as by the addition of bleach containing sodium bicarbonate in the presence of sodium bromide and TEMPO in EtOAc, toluene, and water, to give rise to the ketone. The ketone is converted to the dimethylketal by the addition of trimethyl-orthoformate in the presence of paratoluenesulphonic acid in methanol. Finally, the protecting group is removed by hydrogenation using, for example, palladium an charcoal in the presence of ethanol under an atmosphere of hydrogen.
excessive bone or cartilage loss, including osteoporosis, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease; hypercalcemia of malignancy, and metabolic bone disease.
Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix, and certain tumors and metastatic neoplasias may be effectively treated with the compounds of this invention.
The present invention also provides methods of treatment of diseases caused by pathological levels of proteases, particularly cysteine and serine proteases, more particularly cysteine proteases, even more particularly as inhibitors of cysteine proteases of the papain superfamily, yet more particularly cysteine proteases of the cathepsin family, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of the present invention. The present invention especially provides methods of treatment of diseases caused by pathological levels of cathepsin K, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof, an inhibitor of cathepsin K, including a compound of the present invention. The present invention particularly provides methods for treating diseases in which cysteine proteases are implicated, including infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and especially diseases in which cathepsin K is implicated, most particularly diseases of excessive bone or cartilage loss, including osteoporosis, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease.
This invention further provides a method for treating osteoporosis or inhibiting bone loss which comprises internal administration to a patient of an effective amount of a compound of formula (I), alone or in combination with other inhibitors of bone resorption, such as bisphosphonates (i.e., allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In addition, treatment with a compound of this invention and an anabolic agent, such as bone morphogenic protein, iproflavone, may be used to prevent bone loss or to increase bone mass.
In accordance with this invention, an effective amount of the compounds of formula (I) is administered to inhibit the protease implicated with a particular condition or disease. Of course this dosage amount will further be modified according to the type of administration of the compound. For example, xe2x80x9ceffective amountxe2x80x9d for acute therapy, parenteral administration of a compound of formula (I) is preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit cathepsin K. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
Prodrugs of compounds of the present invention may be prepared by any suitable method. For those compounds in which the prodrug moiety is a ketone functionality, specifically ketals and/or hemiacetals, the conversion may be effected in accordance with conventional methods.
The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The compounds of this invention may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.
Determination of Cathepsin K Proteolytic Catalytic Activity
All assays for cathepsin K were carried out with human recombinant enzyme. Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically Cbz-Phe-Arg-AMC, and were determined in 100 mM Na acetate at pH 5.5 containing 20 mM cysteine and 5 mM EDTA. Stock substrate solutions were prepared at concentrations of 10 or 20 mM in DMSO with 20 xcexcM final substrate concentration in the assays. All assays contained 10% DMSO. Independent experiments found that this level of DMSO had no effect on enzyme activity or kinetic constants. All assays were conducted at ambient temperature. Product fluorescence (excitation at 360 nM; emission at 460 nM) was monitored with a Perceptive Biosystems Cytofluor II fluorescent plate reader. Product progress curves were generated over 20 to 30 minutes following formation of AMC product.
Inhibition Studies
Potential inhibitors were evaluated using the progress curve method. Assays were carried out in the presence of variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of inhibitor and substrate. Data analysis was conducted according to one of two procedures depending on the appearance of the progress curves in the presence of inhibitors. For those compounds whose progress curves were linear, apparent inhibition constants (Ki,app) were calculated according to equation 1 (Brandt et al., Biochemitsry, 1989, 28, 140):
v=VmA/[Ka(1+I/Ki,app)+A]xe2x80x83xe2x80x83(1) 
where v is the velocity of the reaction with maximal velocity Vm, A is the concentration of substrate with Michaelis constant of Ka, and I is the concentration of inhibitor.
For those compounds whose progress curves showed downward curvature characteristic of time-dependent inhibition, the data from individual sets was analyzed to give kobs according to equation 2:
[AMC]=vSSt+(v0xe2x88x92vSS)[1xe2x88x92exp (xe2x88x92kobst)]/kobs xe2x80x83xe2x80x83(2) 
where [AMC] is the concentration of product formed over time t, v0 is the initial reaction velocity, and vSS is the final steady state rate. Values for kobs were then analyzed as a linear function of inhibitor concentration to generate an apparent second order rate constant (kobs/inhibitor concentration or kobs/[I]) describing the time-dependent inhibition. A complete discussion of this kinetic treatment has been fully described (Morrison et al., Adv. Enzymol. Relat. Areas Mol. Biol., 1988, 61, 201).
One skilled in the art would consider any compound with a Ki of less than 50 micromolar to be a potential lead compound. Preferably, the compounds used in the method of the present invention have a Ki value of less than 1 micromolar. Most preferably, said compounds have a Ki value of less than 100 nanomolar. 4-(R,S)-Amino-N-[(8-quinolinesulfonyl)-S-leucine]-3-tetrahydrofuran-3-one, a compound of formula (I), has a Ki value that is greater than 10 micromolar.
Human Osteoclast Resorption Assay
Aliquots of osteoclastoma-derived cell suspensions were removed from liquid nitrogen storage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4xc2x0 C.). The medium was aspirated and replaced with murine anti-HLA-DR antibody, diluted 1:3 in RPMI-1640 medium, and incubated for 30 minutes on ice. The cell suspension was mixed frequently.
The cells were washed xc3x972 with cold RPMI-1640 by centrifugation (1000 rpm, 5 min at 4xc2x0 C.) and then transferred to a sterile 15 mL centrifuge tube. The number of mononuclear cells were enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG, were removed from their stock bottle and placed into 5 mL of fresh medium (this washes away the toxic azide preservative). The medium was removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads were mixed with the cells and the suspension was incubated for 30 minutes on ice. The suspension was mixed frequently. The bead-coated cells were immobilized on a magnet and the remaining cells (osteoclast-rich fraction) were decanted into a sterile 50 mL centrifuge tube. Fresh medium was added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process was repeated xc3x9710. The bead-coated cells were discarded.
The osteoclasts were enumerated in a counting chamber, using a large-bore disposable plastic pasteur pipette to charge the chamber with the sample. The cells were pelleted by centrifugation and the density of osteoclasts adjusted to 1.5xc3x97104/mL in EMEM medium, supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate. 3 mL aliquots of the cell suspension ( per treatment) were decanted into 15 mL centrifuge tubes. These cells were pelleted by centrifugation. To each tube 3 mL of the appropriate treatment was added (diluted to 50 xcexcM in the EMEM medium). Also included were appropriate vehicle controls, a positive control (87MEM1 diluted to 100 ug/mL) and an isotype control (IgG2a diluted to 100 ug/mL). The, tubes were incubated at 37xc2x0 C. for 30 minutes.
0.5 mL aliquots of the cells were seeded onto sterile dentine slices in a 48-well plate and incubated at 37xc2x0 C. for 2 hours. Each treatment was screened in quadruplicate. The slices were washed in six changes of warm PBS (10 mL/well in a 6-well plate) and then placed into fresh treatment or control and incubated at 37xc2x0 C. for 48 hours. The slices were then washed in phosphate buffered saline and fixed in 2% glutaraldehyde (in 0.2M sodium cacodylate) for 5 minutes, following which they were washed in water and incubated in buffer for 5 minutes at 37xc2x0 C. The slices were then washed in cold water and incubated in cold acetate buffer/fast red garnet for 5 minutes at 4xc2x0 C. Excess buffer was aspirated, and the slices were air dried following a wash in water.
The TRAP positive osteoclasts were enumerated by bright-field microscopy and were then removed from the surface of the dentine by sonication. Pit volumes were determined using the Nikon/Lasertec ILM21W confocal microscope.